random primers for qPCR - (May/17/2006 )
Hi to everybody,
I was wandering if random primers can be used for reverse trascription and following real time PCR for quantification of gene expression. Someone has told me that the reaction could loss its linearity because random primers can bind to multiple site and generate multiple transcripts by one RNAm molecule biasing quantification.
Is that true ? Someone has experienced random primers in qPCR ?
Thanks ,
Simone
you can use random primers to make cDNA, but you cannot use them for qPCR
if you used them during the qPCR step, you would get an assortment of products, not from just one RNA, either...but from all the cDNA
for the quantitative part, where the amplification occurs, you must have gene specific primers and they must be very carefully designed...otherwise your results will not be quantitative at all...does this make sense to you?
if you used them during the qPCR step, you would get an assortment of products, not from just one RNA, either...but from all the cDNA
for the quantitative part, where the amplification occurs, you must have gene specific primers and they must be very carefully designed...otherwise your results will not be quantitative at all...does this make sense to you?
Yes this makes sense to me. I was wandering how in a paper I am reading on gene expression analysis they say that they have used random hexamers for cDNA synthesis. Housekeeping genes have been used for relative quantification in this paper, can it make the difference ?
Thanks
go back and read the first sentence of my last post... 